Tài liệu Báo cáo khoa học: Amprenavir complexes with HIV-1 protease and its drug-resistant mutants altering hydrophobic clusters docx

Amprenavir complexes with HIV-1 protease and its drug-resistant mutants altering hydrophobic clusters Chen-Hsiang Shen1, Yuan-Fang Wang1, Andrey Y Kovalevsky1,*, Robert W Harrison1,2 and Irene T Weber1,3 Department of Biology, Molecular Basis of Disease Program, Georgia State University, Atlanta, GA, USA Department of Computer Science, Molecular Basis of Disease Program, Georgia State Univers’ity, Atlanta, GA, USA Department of Chemistry, Molecular Basis of Disease Program, Georgia State University, Atlanta, GA, USA Keywords aspartic protease; conformational change; enzyme inhibition; HIV ⁄ AIDS; X-ray crystallography Correspondence I T Weber, Department of Biology, Georgia State University, PO Box 4010, Atlanta, GA 30302-4010, USA Fax: 404 413 5301 Tel: 404 413 5411 E-mail: iweber@gsu.edu *Present address Bioscience Division, MS M888, Los Alamos National Laboratory, Los Alamos, NM, USA Database The atomic coordinates and structure factors are available in the Protein Data Bank with accession code 3NU3 for wild-type HIV-1 PR–APV, 3NU4 for PRV32I– APV, 3NU5 for PRI50V–APV, 3NU6 for PRI54M–APV, 3NUJ for PRI54V–APV, 3NU9 for PRI84V–APV, and 3NUO for PRL90M–APV (Received 23 March 2010, revised 25 June 2010, accepted 12 July 2010) The structural and kinetic effects of amprenavir (APV), a clinical HIV protease (PR) inhibitor, were analyzed with wild-type enzyme and mutants with single substitutions of V32I, I50V, I54V, I54M, I84V and L90M that are common in drug resistance Crystal structures of the APV complexes at ˚ resolutions of 1.02–1.85 A reveal the structural changes due to the mutations Substitution of the larger side chains in PRV32I, PRI54M and PRL90M resulted in the formation of new hydrophobic contacts with flap residues, residues 79 and 80, and Asp25, respectively Mutation to smaller side chains eliminated hydrophobic interactions in the PRI50V and PRI54V structures The PRI84V–APV complex had lost hydrophobic contacts with APV, the PRV32I–APV complex showed increased hydrophobic contacts within the hydrophobic cluster and the PRI50V complex had weaker polar and hydrophobic interactions with APV The observed structural changes in PRI84V–APV, PRV32I–APV and PRI50V–APV were related to their reduced inhibition by APV of six-, 10- and 30-fold, respectively, relative to wildtype PR The APV complexes were compared with the corresponding saquinavir complexes The PR dimers had distinct rearrangements of the flaps and 80¢s loops that adapt to the different P1¢ groups of the inhibitors, while maintaining contacts within the hydrophobic cluster These small changes in the loops and weak internal interactions produce the different patterns of resistant mutations for the two drugs Structured digital abstract l MINT-7966480: HIV-1 PR (uniprotkb:P03366) and HIV-1 PR (uniprotkb:P03366) bind (MI:0407) by x-ray crystallography (MI:0114) doi:10.1111/j.1742-4658.2010.07771.x Introduction Currently,  33 million people worldwide are estimated to be infected with HIV in the AIDS pandemic [1] The virus cannot be fully eradicated, despite the effectiveness of highly active antiretroviral therapy [2] Furthermore, the development of vaccines has been extremely challenging [3] Highly active antiretroviral Abbreviations APV, amprenavir; DPI, diffraction data precision indicator; DRV, darunavir; MES, 2-(N-morpholino)ethanesulfonic acid; PI, HIV-1 protease inhibitor; PR, HIV-1 protease; PRWT, wild-type PR; PRV32I, PR with the V32I mutation; PRI50V, PR with the I50V mutation; PRI54M, PR with the I54M mutation; PRI54V, PR with the I54V mutation; PRI84V, PR with the I84V mutation; PRL90M, PR with the L90M mutation; SQV, saquinavir; THF, tetrahydrafuran FEBS Journal 277 (2010) 3699–3714 ª 2010 The Authors Journal compilation ª 2010 FEBS 3699 HIV protease mutants altering hydrophobic clusters C-H Shen et al therapy uses more than 20 different drugs, including inhibitors of the HIV-1 enzymes, reverse transcriptase, protease (PR) and integrase, as well as inhibitors of cell entry and fusion The major challenge limiting current therapy is the rapid evolution of drug resistance due to the high mutation rate caused by the absence of a proof-reading function in HIV reverse transcriptase [4] PR is the enzyme responsible for the cleavage of the viral Gag and Gag-Pol polyproteins into mature, functional proteins PR is a valuable drug target, as inhibition of PR activity results in immature noninfectious virions [5,6] PR is a dimeric aspartic protease composed of residues 1–99 and 1¢–99¢ The conserved catalytic triplets, Asp25-Thr26-Gly27, from both subunits provide the key elements for formation of the enzyme active site Inhibitors and substrates bind in the active site cavity between the catalytic residues and the flexible flaps comprising residues 45–55 and 45¢–55¢ [7] Amprenavir (APV) was the first PR inhibitor (PI) to include a sulfonamide group (Fig 1A) Similar to other PIs, APV contains a hydroxyethylamine core that mimics the transition state of the enzyme Unlike the first generation PIs, such as saquinavir (SQV), APV was designed to maximize hydrophilic interactions with PR [8] The sulfonamide group increases the water solubility of APV (60 lgỈmL)1) compared with SQV (36 lgỈmL)1) [9] The crystal structures of PR complexes with APV [8,10] and SQV [11,12] demonstrated the critical PR–PI interactions HIV-1 resistance to PIs arises mainly from the accumulation of PR mutations Conservative mutations of hydrophobic residues are common in PI resistance, including V32I, I50V, I54V ⁄ M, I84V and L90M, which are the focus of this study [13] The location of these mutations in the PR dimer structure is shown in Fig 1B Multidrug-resistant mutation V32I, which alters a residue in the active site cavity, appears in  20% of patients treated with APV [14] and is associated with high levels of drug resistance to lopinavir ⁄ ritonavir [13] Ile50 and Ile54 are located in the flap region, which is important for catalysis and binding of substrates or inhibitors [8,15] Mutations of flap residues can alter the protein stability or binding of inhibitors [15–18] PR with mutation I50V shows nine-fold worse inhibition by darunavir (DRV) relative to wildtype enzyme [19], and 50- and 20-fold decreased inhibition by indinavir and SQV [17,18] Unlike Ile50, Ile54 does not directly interact with APV, but mutations of Ile54 are frequent in APV resistance and the I54M mutation causes six-fold increased IC50 [20] Mutation I54V appears in resistance to indinavir, lopinavir, nelfinavir and SQV [13] I54V in combination with other 3700 mutations, especially V82A [21,22], decreases the susceptibility to PI therapy [18] I84V, which is located in the active site cavity, significantly reduces drug susceptibility to APV [23] L90M is commonly found during PI treatment [14] and is resistant to all currently used PIs, with major effects on nelfinavir and SQV [13] Mutations of hydrophobic residues are found in more than half of drug-resistant mutants [13,24] and several of these mutations show altered PR stability [17,25] Hydrophobic interactions play an important role in protein stability Aliphatic groups reportedly contribute  70% of the hydrophobic interactions in proteins [26] Removing a methyl group in the protein hydrophobic core affects protein folding and decreases the protein stability in mutant proteins [27] In PR, two clusters of methyl groups have been identified; one inner cluster surrounding the active site cavity and the second cluster in an outer hydrophobic core, as shown in Fig 1B [24] Drug-resistant mutations V32I, I50V, I54V ⁄ M and I84V belong to the inner cluster around the active site, whereas L90M is in the outer cluster In order to establish a better understanding of the mechanism of resistance to APV, atomic and high-resolution crystal structures have been determined of APV complexes with wild-type PR (PRWT) and its mutants containing single substitutions of Val32, Ile50, Ile54, Ile84 and Leu90 PR mutations can have distinct effects on the binding of different inhibitors Therefore, the structural effects of APV and SQV were compared for PRWT and mutants PRI50V, PRI54M and PRI54V complexes, using previously reported SQV complexes [12,18] Exploring the changes in PR due to binding of two different inhibitors will give insight into the mechanisms of resistance and help in the design of new inhibitors Results APV inhibition of PR and mutants The kinetic parameters and inhibition constants of APV for PRWT and the drug-resistant mutants PRV32I, PRI50V, PRI54M, PRI54V, PRI84V and PRL90M are shown in Table The lowest catalytic efficiency (kcat ⁄ Km) values were seen for PRV32I and PRI50V, with 30% and 10% of the PRWT value, respectively PRL90M showed a surprisingly high 11-fold increase in catalytic efficiency, whereas the other mutants were similar to PRWT The kcat ⁄ Km values for PRL90M appear to depend on the substrate, however, as only a modest three-fold increase relative to PRWT was observed using a different substrate with the sequence derived from the MA ⁄ CA rather than the p2 ⁄ NC cleavage site [19] The six mutants and PRWT FEBS Journal 277 (2010) 3699–3714 ª 2010 The Authors Journal compilation ª 2010 FEBS C-H Shen et al HIV protease mutants altering hydrophobic clusters A P2′ P1 P2 O P1 NH2 O O S N N H O OH H N O HO O P3 P1′ N NH2 P2 Amprenavir B O O H N N O P2′ H N P1′ Saquinavir 54 47 50 77 76 Fig (A) The chemical structures of APV and SQV (B) The structure of PR dimer with the sites of mutation Val32, Ile50, Ile54, Ile84 and Leu90 indicated by green sticks for side chain atoms in both subunits Amino acids are labeled in one subunit only APV is shown in magenta sticks The amino acids in the inner hydrophobic cluster are indicated by numbered red spheres, and the amino acids in the outer hydrophobic cluster are shown as blue spheres 75 89 66 90 23 22 15 26 24 38 33 84 28 85 71 36 82 32 62 56 80 12 11 13 64 93 Table Kinetic parameters for substrate hydrolysis and inhibition of APV The error in kcat ⁄ Km is calculated as (A ⁄ B) ± (1 ⁄ B2)[square root (B2a2 + A2b2)], where A is kcat, a is kcat error, B is Km and b is error in Km Km (lM) a WT V32I I50Va I54Ma I54Va I84V L90M kcat (min)1) kcat ⁄ Km (lMỈmin)1) Relative kcat ⁄ Km Ki (nM) 30 65 109 41 43 73 13 190 120 68 300 130 320 950 6.5 1.8 0.6 7.3 3.1 4.4 73 1.0 0.3 0.1 1.1 0.5 0.7 11.2 0.15 1.5 4.5 0.50 0.41 0.9 0.16 ± ± ± ± ± ± ± 6 ± ± ± ± ± ± ± 20 10 40 20 30 120 ± ± ± ± ± ± ± 1.3 0.2 0.03 0.8 0.9 0.5 13 ± ± ± ± ± ± ± Relative Ki 0.04 0.2 0.6 0.06 0.05 0.2 0.01 10 30 3 a Km and kcat values from [18] were assayed for inhibition by APV (Table 1) APV showed subnanomolar inhibition with a Ki of 0.16 nm for PRWT and PRL90M PRI54M and PRI54V showed modestly increased (three-fold) relative Ki values The largest increases in Ki of six-, 10- and 30-fold were observed for PRI84V, PRV32I and PRI50V, respectively, relative to PRWT The substantially decreased inhibition of PRV32I and PRI50V suggested the loss of interactions with APV Crystal structures of APV complexes The crystal structures of PR and drug-resistant mutants PRV32I, PRI50V, PRI54M, PRI54V, PRI84V and PRL90M were determined in their complexes with APV ˚ at resolutions of 1.02–1.85 A to investigate the structural changes The crystallographic data are summarized in Table All structures were determined in space group P21212 The asymmetric unit contains one PR dimer of residues 1–99 and 1¢–99¢ as well as APV The lowest resolution structure of PRI84V was refined to an R-factor of 0.20 with isotropic B-factors and solvent molecules The other structures were refined at ˚ 1.50–1.02 A resolution to R-factors of 0.12–0.16, including anisotropic B-factors, hydrogen atoms and solvent molecules PRWT had the highest resolution and lowest R-factor, concomitant with the lowest average B-factors for the protein and inhibitor atoms Because of the high resolution of the diffraction data, all structures except for PRI84V–APV, were FEBS Journal 277 (2010) 3699–3714 ª 2010 The Authors Journal compilation ª 2010 FEBS 3701 HIV protease mutants altering hydrophobic clusters C-H Shen et al Table Crystallographic data collection and refinement statistics All were refined with SHELX-97, except PRI84V, which was refined using REFMAC 5.2 Values in parentheses are given for the highest resolution shell Rmerge = Rhkl|Ihkl ) ÆIhklæ| ⁄ RhklIhkl; R = R|Fobs ) Fcal| ⁄ RFobs; Rfree = Rtest(|Fobs| ) |Fcal|)2 ⁄ Rtest|Fobs|2 APV complex PR PRV32I PRI50V PRI54M PRI54V PRI84V PRL90M Space group P21212 P21212 P21212 P21212 P21212 P21212 P21212 ˚ Unit cell dimensions: (A) A B C ˚ Resolution range (A) Unique reflections Rmerge (%) overall I ⁄ r(I) overall Completeness (%) overall ˚ Data range for refinement (A) R (%) Rfree (%) No of solvent atoms (total occupancies) 58.11 85.97 46.42 50–1.02 113 227 5.7 (38.2) 15.3 (2.6) 95.8 (65.0) 10–1.02 12.4 14.2 292 (207.3) 57.77 86.13 46.28 50–1.20 66 626 8.1 (44.2) 11.3 (2.5) 91.6 (62.7) 10–1.20 16.4 20.1 151 (129.8) 57.95 86.01 46.21 50–1.29 55 569 7.0 (40.2) 15.2 (2.3) 93.9 (70.4) 10–1.29 15.5 19.3 177 (143.6) 58.12 85.91 46.10 50–1.16 73 638 7.2 (35.7) 20.1 (2.1) 91.8 (58.9) 10–1.16 15.4 18.8 242 (221.5) 57.50 86.00 45.95 50–1.50 37 010 6.0 (46.2) 16.8 (2.4) 99.7 (99.2) 10–1.50 14.9 19.7 152 (128.5) 59.51 86.88 45.44 50–1.85 18 138 9.7 (34.5) 15.8 (5.8) 93.2 (76.6) 10–1.85 19.9 23.6 84 (84) 57.94 85.91 46.10 50–1.35 50 443 5.5 (46.2) 17.9 (2.5) 97.8 (97.3) 10–1.35 14.3 19.9 211 (202.5) RMS deviation from ideality ˚ Bonds (A) ˚ Angle distance (A) 0.017 0.036 0.013 0.031 0.012 0.030 0.016 0.033 0.010 0.029 0.014 1.546 (Degree) 0.012 0.030 ˚ Average B-factors (A2) Main chain atoms Side chain atoms Inhibitor Solvent DPI Relative occupancy of APV ˚ RMS deviation from PR (A) ˚ RMS deviation from PR–SQV (A) 10.8 14.8 10.5 20.8 0.02 0.7 ⁄ 0.3 – 0.87 16.0 21.4 16.9 25.6 0.04 – 0.15 – 14.4 20.7 17.1 24.3 0.05 0.6 ⁄ 0.4 0.19 0.29 14.2 20.8 17.8 36.1 0.04 – 0.33 0.36 23.2 28.8 28.5 47.0 0.07 – 0.26 0.32 25.6 28.3 23.7 49.5 0.13 – 0.38 0.36 20.3 23.6 16.1 39.9 0.05 – 0.19 – modeled with more than 150 water molecules, ions and other small molecules from the crystallization solutions, including many with partial occupancy (Table 2) The solvent molecules were identified by the shape and intensity of the electron density and the potential for interactions with other molecules The nonwater-solvent molecules were: a single sodium ion, three chloride ions, two partial glycerol molecules in PRWT–APV; one sodium ion, three chloride ions in PRV32I–APV; three sodium ions, seven chloride ions, two partial acetate ions in PRI50V–APV; one sodium ions, three chloride, two partial acetate ions in PRI54M–APV; 19 iodide ions in PRI54V–APV; 33 iodide ions in PRI84V–APV; and 19 iodide ions in PRL90M– APV However, many iodide ions had partial occupancy They were identified by the high peaks in electron density maps, abnormal B-factors and contact ˚ distances of 3.4–3.8 A to nitrogen atoms Alternative conformations were modeled for residues in all crystal structures Alternative conformations were modeled for a total of 48, 13, 28, 11, 1, residues in PRWT–APV, PRV32I–APV, PRI50V–APV, PRI54M– 3702 APV, PRI54V–APV, PRI84V–APV and PRL90M–APV structures, respectively APV was observed in two alternative orientations related by a rotation of 180° in the complexes with PRWT and PRI50V with relative occupancies of 0.7 ⁄ 0.3 and 0.6 ⁄ 0.4, respectively The highest resolution structure, PRWT–APV, showed the most alternative conformations for main chain and side chain residues Several residues in the active site cavity showed two alternative conformations and were refined with the same relative occupancies as for APV Surface residues with longer flexible side chains, such as Trp6, Arg8, Glu21, Glu34, Ser37, Lys45, Met46, Lys55, Arg57, Gln61 and Glu65, were refined with alternative conformations Also, some internal hydrophobic residues, such as Ile64, Leu97, showed a second conformation for the side chain At the other extreme, the lowest resolution structure of PRI84V–APV showed only one residue, Leu97, with an alternative side chain conformation In all the structures, the two catalytic Asp25 residues showed negative difference density around the carboxylate oxygens This phenomenon might be caused by radiation damage in the carboxylate FEBS Journal 277 (2010) 3699–3714 ª 2010 The Authors Journal compilation ª 2010 FEBS C-H Shen et al HIV protease mutants altering hydrophobic clusters side chains, especially due to their location at the active site, as described in [28] The accuracy in the atomic positions was evaluated by the diffraction data precision indicator (DPI), which is calculated in sfcheck from the resolution, R-factor, completeness and observed data [29] The highest resolution structure of PRWT–APV had the ˚ lowest DPI value of 0.02 A, whereas the lowest resolution structure of PRI84V–APV had the highest DPI ˚ value of 0.13 A (Table 2) We estimate that significant differences in interatomic distances should be at least three-fold larger than the DPI value [7] Hence, struc˚ tural changes > 0.06 A are significant for PRWT–APV ˚ for PRI84V–APV at the two extremes of and > 0.4 A resolution The quality of the crystal structures is illustrated by the 2Fo–Fc electron density maps for the mutated residues (Fig S1) The mutated residues had single conformations, except for the side chains of Met54, Val54 and Met90 in one subunit that were refined with relative occupancies of 0.6 ⁄ 0.4, 0.7 ⁄ 0.3 A and 0.5 ⁄ 0.5, respectively Overall, the mutants and wild-type enzyme had very similar structures, probably because they shared the same crystallographic unit cell The PRI54M, PRI54V and PRI84V complexes had RMS deviations for the Ca atoms ranging from 0.26 to ˚ 0.38 A compared with the wild-type structure The structures of PRV32I, PRI50V and PRL90M were more ˚ similar to PRWT with RMS deviations of 0.15–0.19 A for the main chain atoms PR interactions with APV and the influence of alternative conformations The atomic resolution crystal structure of PRWT–APV was refined with two differently populated conformations for the inhibitor and several residues forming the binding site with relative occupancies of 0.7 ⁄ 0.3 (Fig 2A) Residues Arg8, Asp30, Val32, Lys45, Gly48, Ile50 and Pro81 showed alternative conformations in both subunits, and Asp25¢ had two alternative Gly48′ Gly48 H 2O Amprenavir Asp30′ Asp30 Asp25 Ile50 B Fig Inhibitor binding site in PRWT–APV (A) APV and PR residues in the binding site with alternative conformations Omit maps for major (green) and minor (magenta) conformations of APV, interacting PR residues Asp25, Gly48 and Asp30 from both subunits, and the conserved flap water are contoured at a level of 3.5 r (B) Hydrogen bond, C-HỈỈỈO and H2ỈỈp interactions between PR (gray) and APV (cyan) Hydrogen bond interactions are indicated by dashed lines C-HỈỈỈO and H2ỈỈp interactions are indicated by dotted lines Asp25′ Gly48′ 2.9 3.0 A 3.6 H2O 2.8 2.9 3.3 3.1 3.2 Ile50′ 3.8 3.1 Asp30′ 3.1 3.5 2.6 Asp29′ Gly48 3.2 Gly27′ FEBS Journal 277 (2010) 3699–3714 ª 2010 The Authors Journal compilation ª 2010 FEBS 2.7 3.0 3.5 H2OB 3.1 APV 3.0 3.1 2.8 H2OC 2.6 3.2 Gly27 Asp29 Asp30 Asp25′ Asp25 3703 HIV protease mutants altering hydrophobic clusters C-H Shen et al conformations for the side chain Alternative conformations were also refined for the main chain of residues 24¢, 29¢, 30, 30¢, 31, 31¢, 48, 48¢, 79¢ and 80¢ around the inhibitor binding site Moreover, the conserved water molecule between the flaps and the inhibitor showed two alternative positions Similar, although less extensive, disorder in the inhibitor binding site has been observed in other atomic resolution crystal structures of this enzyme [12,30] In fact, the highest resolu˚ tion structure reported to date (0.84 A) of PRV32I with DRV comprised two distinct populations for the entire dimer with inhibitor and one conformer contained an unusual second binding site for DRV [30] Moreover, a similar asymmetric arrangement of Asp25 ⁄ 25¢ with a single conformation for Asp25 and two conformations for Asp25¢ was observed in the crystal structure of PRWT–GRL0255A [31] Only single conformations were apparent for APV in the mutant protease structures, with the exception of PRI50V However, the mutant structures were refined with lower resolution data where alternative conformations may be less clearly resolved than for the PRWT–APV structure APV interactions with PRWT were analyzed in terms of the hydrogen bond, C-HỈỈỈO and H2ỈỈp interactions, as described for the PRV32I complex with DRV [30] The polar interactions of the major conformation of APV with PRWT are illustrated in Fig 2B The central hydroxyl group of APV formed strong hydrogen bond interactions with the carboxylate oxygens of the catalytic residues Asp25 and Asp25¢ APV formed four direct hydrogen bonds with the main chain amide of Asp30¢, the carbonyl oxygen of Gly27¢, and the amide and carbonyl oxygen of Asp30 Water molecules make important contributions to the binding site The flap water molecule (H2OA in Fig 2B), which is conserved in almost all PR–inhibitor complexes, formed a tetrahedral arrangement of hydrogen bonds connecting the amide nitrogen atoms of Ile50 ⁄ 50¢ in the flap region with the sulfonamide oxygen and the carbamate carbonyl oxygen of APV The second conserved water (H2OB) bridged APV and the PR main chain by hydrogen bonds to the carbonyl of Gly27 and the amide of Asp29 and a H2ỈỈp interaction with the aniline group of APV The interactions of H2OB are conserved in PR complexes with DRV and antiviral inhibitors based on the same chemical scaffold [31,32] The third water, H2OC, which is conserved in these APV complexes and in DRV complexes, mediated hydrogen bond interactions between the carboxylate of Asp30 and aniline NH2 of APV Also, several C-HỈỈỈO interactions linked the PR main chain to APV: the carbonyl oxygens of Gly48¢ and Asp30¢ interacted with the tetrahydrafuran (THF) moiety, Gly27 carbonyl 3704 oxygen with the isopropyl group, Gly48 carbonyl oxygen with the aniline ring, the sulfonamide oxygens of APV to Gly49 and Ile50¢, APV carbonyl oxygen with Gly49Â and APV oxygen to Gly27Â (Fig 2) The CHặặặO interactions formed by the PR amides and carbonyl oxygens mimic the conserved hydrogen bond interactions observed in PR complexes with peptide analogs [33,34] The minor APV conformation refined with 0.3 relative occupancy lay in the opposite orientation to the major conformation and interacted with the opposite subunits of the PR dimer The minor conformation of APV retained almost identical hydrogen bond, C-HỈỈỈO and H2ỈỈp interactions to the major conformation, with the following exceptions (Table S1) The hydrogen bond between the aniline nitrogen of APV and the carbonyl oxygen of Asp30 was lost in the minor APV ˚ conformation (distance increased to 3.6 A) The watermediated interaction between the APV aniline nitrogen and the carboxylate group of Asp30 was replaced by a ˚ weak direct hydrogen bond (distance of 3.4 A) The hydrogen bond of the THF oxygen with the amide of Asp30¢ was lost in the minor APV conformation (dis˚ tance increased to 3.7 A) Instead, the THF oxygen of APV formed a new interaction with the carboxylate group in the minor conformation of Asp30 The water interaction with the amide of Ile50¢ was weakened (dis˚ tance of 3.5 A) The C-HỈỈỈO interaction between the carbonyl oxygen of APV and the Ca of Gly49 was lost in the minor conformation of APV Some of these differences probably reflect the lower occupancy and greater positional error in the minor conformation Variability in the interactions of Asp30 ⁄ 30¢ due to flexibility of the side chains has been observed in other PR complexes [19] Overall, the minor conformation of APV showed one less hydrogen bond, one less C-HỈỈỈO interaction and weaker interactions than the major conformation had with PR Effects of mutations on PR structure and interactions with APV The structures of the mutants and PRWT complexes with APV were compared in order to identify any significant changes Overall, the polar interactions between APV and PR were well maintained in the mutant complexes In these seven complexes the distances between nonhydrogen atoms were observed to ˚ be in the range of 2.3–3.3 A for hydrogen bonds and ˚ 3.2–3.8 A for C-HỈỈỈO interactions (Tables S1 and S2) ˚ The estimated error in atomic position is  0.05 A in ˚ structures at 1.0–1.2 A resolution compared with the ˚ higher estimated errors of 0.10–0.15 A in structures at FEBS Journal 277 (2010) 3699–3714 ª 2010 The Authors Journal compilation ª 2010 FEBS C-H Shen et al ˚ 1.5–1.8 A resolution [7], such as the complexes of PRI54V–APV and PRI84V–APV Structural changes are detailed below for the mutant complexes with respect to the major conformation in PRWT–APV Generally, the changes in the mutants involved hydrophobic C-HỈỈỈH-C contacts or C-HỈỈỈO polar interactions, although shifts of main chain atoms were observed in some cases The ideal distances between nonhydrogen ˚ atoms are considered to be 3.0–3.7 A for C-HỈỈỈO inter˚ for van der Waals interactions, actions and 3.8–4.2 A as described in [30] The structural differences are described separately for each mutant Val32 is an important part of the S2 pocket in the active site cavity and forms van der Waals interactions with inhibitors In the PRWT–APV structure, Val32 forms hydrophobic contacts with Ile47, Ile56, Thr80 and Ile84, whereas Val32¢ interacts with Thr80¢ and Ile84¢ Mutation of Val to Ile, which adds one methyl group, can reduce the volume of the active site cavity and alter the hydrophobic interactions in the cluster HIV protease mutants altering hydrophobic clusters The mutant with Ile32 did not show significant alterations in the main chain conformation or the interactions with APV However, the Cd1 methyl of the Ile side chain provided new van der Waals contacts with other hydrophobic side chains Ile32 formed new hydrophobic contacts with the side chains of Val56, Leu76 and the main chain atoms of residues 77–78, and Ile32¢ showed new interactions with the side chains of Ile47¢, Ile50 and Val56¢ in the flaps (Fig 3A) The flaps can exist in an open conformation in the absence of inhibitor and a closed conformation when inhibitor is bound The interactions of residue 32 differ in the closed and open conformations; Val32 has no hydrophobic contacts with flap residues in the PR–APV structure, whereas Val32 forms hydrophobic contacts with Ile47 in the open conformation structure [35] The flexibility of the flaps is probably altered in PRV32I by the new hydrophobic contacts of Ile32 ⁄ 32¢, which is expected to contribute to the three-fold reduced catalytic activity and the 10-fold decreased APV inhibition Fig The interactions of mutated residues in (A) PRV32I–APV, (B) PRI50V–APV, (C) PRI54M–APV, (D) PRI54V–APV, (E) PRI84V–APV and (F) PRL190M–APV The gray color corresponds to PRLWT–APV and the cyan color indicates the mutant complex Dashed lines indicate van ˚ der Waals interactions and dotted lines show C-HỈỈỈO interactions Interatomic distances are shown in A with black lines indicating PRWT and ˚ red lines indicating the mutant Interatomic distances of > 4.3 A are shown in dash-dot lines to indicate the absence of favorable interaction FEBS Journal 277 (2010) 3699–3714 ª 2010 The Authors Journal compilation ª 2010 FEBS 3705 HIV protease mutants altering hydrophobic clusters C-H Shen et al of the PRV32I mutant relative to wild-type enzyme (Table 1) Ile50 is located at the tip of the flap on each PR monomer, where its side chain forms hydrophobic interactions with inhibitors In the wild-type enzyme, Ile50 ⁄ Ile50¢ interacts with Pro81¢ ⁄ Pro81 and Thr80¢ ⁄ Thr80 in the 80¢s loop, as well as Ile47¢ ⁄ 47 and Ile54¢ ⁄ 54 in the flaps The Cd1 methyl of the Ile50 side chain forms C-HỈỈỈO interactions with the hydroxyl oxygen of Thr80¢ and carbonyl oxygen of Pro79¢, and the Cd1 of Ile50¢ interacts with the hydroxyl of Thr80 Mutation from Ile50 to Val shortens the side chain by a methyl group, which eliminates the C-HỈỈỈO interaction with the hydroxyl oxygen of Thr80¢ and van der Waals contact with Ile54¢ (Fig 3B) In the other subunit, mutation to Val50Â eliminates the C-HặặặO interaction with the hydroxyl of Thr80 and a hydrophobic contact with Pro81 The APV in PRI50V complex had two alternative conformations with 0.6 ⁄ 0.4 relative occupancy The APV showed a more elongated hydrogen bond than seen in the PRWT complex between the aniline group and the carbonyl oxygen of ˚ Asp30, with an interatomic distance of 3.4 A for the ˚ for the minor APV major conformation and 3.5 A conformation Val50¢ also lost hydrophobic interactions with the THF group of APV The minor conformation of APV showed similar changes in interactions with Asp30 ⁄ 30¢ as described for the minor APV conformation in the PRWT complex Overall, the observed structural changes in PRI50V–APV were the loss of two C-HỈỈỈO interactions and van der Waals contacts, the elongated hydrogen bond and reduced hydrophobic contacts with APV PRI50V showed a large decrease in sensitivity to APV shown by the 30-fold drop in the relative inhibition coupled with 10-fold decreased catalytic efficiency, which suggests the importance of Ile50 Loss of the C-HỈỈỈO interaction of Val50 with Thr80¢ has not been previously described Thr80 is a conserved residue in the PR sequences and its hydroxyl forms a hydrogen bond with the carbonyl oxygen of Val82, which contributes hydrophobic interactions with the inhibitors Moreover, the hydroxyl group of Thr80 was shown to be important for PR activity using site-directed mutagenesis where only mutation to Ser retaining the hydroxyl group, and not to Val or Asn, maintained enzymatic activity [36] These lines of evidence, taken together, strengthen the suggestion that loss of the C-HỈỈỈO interaction of residue 50 with the hydroxyl of Thr80, as well as loss of hydrophobic contacts with inhibitor, are important for the decreased catalytic activity and APV inhibition of PRI50V Ile54 is another flap residue that forms hydrophobic interactions with Ile50¢ and residues 79–80, although it 3706 has no direct contact with inhibitor Mutation I54M introduces a longer side chain, and the nearby main chain atoms have shifted relative to their positions in PRWT (Fig 3C) Compared with PRWT, the Ca of ˚ Met54 moved by 0.7 A, and the longer Met side chain ˚ pushed residues 79, 80 and 81 away by 0.7–1.4 A In ˚ the other subunit, the Ca of Met54¢ moved by 0.5 A towards Pro79¢, and there was a correlated motion of ˚ Pro79¢ of 0.9 A relative to its position in PR–APV The longer Met54 ⁄ 54¢ side chains formed more hydrophobic contacts with Pro79 ⁄ 79¢ and Thr80 ⁄ 80¢ in PRI54M relative to those of PRWT Overall, the Ile54 to Met mutation improved contacts within the hydrophobic cluster, although the interatomic distances to residues 79–80 ⁄ 79¢–80¢ were increased Similar structural changes were observed in the PRI54M complexes with DRV and SQV [18] Despite these correlated changes between the main chain atoms of the flaps and 80¢ loops, this mutant was similar to PRWT in catalytic efficiency and had only three-fold reduced inhibition by APV In contrast to PRI54M, mutation I54V substitutes the shorter Val in PRI54V In PRWT, the Cd1 of Ile54 interacted with Ile50¢, Val56, Pro79 and Thr80, whereas the Cd1 of Ile54¢ showed van der Waals interactions with Ile47¢, Ile50¢, Val56¢ and Pro79 and one C-HặặặO interaction with the carbonyl oxygen of Pro79Â The shorter Val side chain in the mutant resulted in loss of several van der Waals contacts with the adjacent residues, thus decreasing the stability of the hydrophobic cluster formed by flap residues 47, 54 and 50¢ (Fig 3D) No C-HặặặO interaction was possible with Pro79Â, which was associated with a shift of  0.5 A in Pro79¢ increasing the separation of the flap and 80¢s loop The mutation I54V decreased the hydrophobic interactions within the flaps and with Pro79 However, PRI54V showed similar Ki value and only a three-fold reduced activity relative to the wild-type enzyme Ile84 forms part of the S1 ⁄ S1¢ subsites of PR, and mutation to Val84 removes a methylene moiety, which can reduce interactions with substrates and inhibitors In PRWT, van der Waals contacts were found between Cd1 of Ile84 and the benzyl and aniline moieties of APV and from Cd1 of Ile84¢ to the isopropyl group of APV These interactions were lost in the PRI84V mutant structure as the interatomic distances increased ˚ to > 4.3 A (Fig 3E) The loss of hydrophobic contacts with APV is consistent with the modest change of six-fold in Ki value for PRI84V Leu90 is located in the short alpha helix outside of the active site cavity, although it extends close to the main chain of the catalytic Asp25 Mutation of Leu90 to Met substituted a longer side chain and introduced FEBS Journal 277 (2010) 3699–3714 ª 2010 The Authors Journal compilation ª 2010 FEBS C-H Shen et al new van der Waals contacts with residues Asp25Thr26 Moreover, the long Met90 ⁄ 90¢ side chains formed close C-HỈỈỈO interactions with the carbonyl oxygen of the catalytic Asp25 and Asp25¢ (Fig 3F) The alternative conformations of the Met90 side chain were arranged as described previously [25] The new interactions of Met90 ⁄ 90¢ with the catalytic aspartates and adjacent residues are presumed to play an important role in the observed 11-fold increase in catalytic activity, as described previously [19,25] The increased catalytic efficiency of the PRL90M mutant is mainly due to an almost five-fold higher kcat On the other hand, the Km of the mutant is only about half that of the wild-type enzyme Therefore, the new interactions of Met90 with the catalytic residues that are absent in the wild-type structure may minimally affect the binding of substrate, but at the same time dramatically lower the activation barrier for substrate hydrolysis, leading to substantial improvement of the PRL90M catalytic activity No change, however, was detected in the APV inhibition of PRL90M Comparison of the mutant complexes with APV and SQV The structures of PR complexes with APV or SQV were analyzed in order to understand their distinct drug resistance profiles PRWT–APV was compared ˚ with PRWT–SQV (2NMW) solved at 1.16 A resolution in a different unit cell and space group P212121 [12] The mutant APV complexes reported here were compared with the published SQV complexes of PRI50V– SQV (3CYX), PRI54M–SQV (3D1X), PRI54V–SQV (3D1Y) and PRI84V–SQV (2NNK) refined at resolu˚ tions of 1.05–1.25 A in the isomorphous unit cell and identical space group P21212 as for all the APV complexes [12,18] No SQV complexes have been reported for mutants PRV32I and PRL90M A lower resolution ˚ (2.6 A) crystal structure has been reported for the SQV complex with the double mutant PRG48V ⁄ L90M [37], in which Met90 showed interactions similar to those seen in the structure of PRL90M–APV To analyze how the PR conformation alters to fit the different inhibitors, all structures were superimposed on the PRWT–APV structure The superposition was tested for both possible arrangements of the two subunits in the asymmetric dimer of PR, i.e superimposing residues 1–99 and 1¢–99¢ with 1–99 and 1¢–99¢, as well as with the opposite subunit arrangement of 1¢–99¢ and 1–99 The arrangement with the lowest RMS deviation was used in further comparison Interestingly, the major conformation of SQV has the opposite orientation to that of APV for the superimposed dimers with HIV protease mutants altering hydrophobic clusters the lowest RMS values PRWT–SQV had the highest ˚ RMS deviation value of 0.87 A on Ca atoms due to the different space groups, whereas the RMS deviations for the mutant complexes with SQV were lower, ˚ ranging from 0.29 to 0.36 A, as usual for two structures in the same space group (Table 2) The corresponding pairs of wild-type and mutant complexes with the two inhibitors were compared (Fig 4A) The structures of PRWT–SQV and PRWT– ˚ APV showed larger RMS deviations of > 1.0 A for residues in surface loops, probably arising from altered lattice contacts due to the different space groups, as reported previously [25] Moreover, the two subunits in the dimer showed asymmetric deviations due to nonidentical lattice contacts as well as the presence of different asymmetric inhibitors Changes in residues 52¢–56¢, 79¢–81¢ in the active site cavity are assumed to reflect variation in the interactions with the two inhibitors, whereas the lower deviations of catalytic triplet residues 25–27 ⁄ 25¢–27¢ reflect their important function The pairs of mutant complexes were determined in isomorphous unit cells with less overall variation so that changes are more likely to arise from different interactions with APV and SQV PRI50V had the fewest RMS differences between the two inhibitor complexes with a ˚ peak of 1.3 A for Phe53¢ PRI54V and PRI84V showed ˚ the largest change for Pro81¢ of 1.9 and 1.6 A, respectively, whereas PRI54M showed the maximum RMS ˚ deviation of 1.2 A at residue 54¢ Three regions were analyzed in more detail due to their flexibility and proximity to inhibitors and mutations: the flaps, the 80¢s loops and the hydrophobic clusters formed by residues Ile47, Ile54, Thr80, Ile84 and Ile50¢ from the opposite subunit The conformation of the flaps segregated into two categories corresponding to the APV complexes and the SQV complexes (Fig 4B) The coordinated changes in the flaps were most obvious for residues 50–51 and 50¢–51¢ at the tips of the flaps The flap residues 50 ˚ and 51 showed differences in Ca position of 0.5–0.9 A between the complexes with APV or SQV Differences ˚ ˚ of 0.6–0.8 A at Gly51¢ and 0.1–0.4 A at Ile ⁄ Val50¢ were seen in the flap from the other subunit The different flap conformations are probably related to the larger chemical groups at P2 and P1¢ in SQV compared with those in APV (Fig 1) Changes in the 80¢s loops, which have been described as intrinsically flexible [12,25,38] and function in substrate recognition [39,40], were assessed using the distance between the Ca atoms of Pro81 and Pro81¢ to reflect alterations in the S1 ⁄ S1¢ subsites ˚ Pro81 and Pro81¢ were separated by 17.6–19.4 A in the APV complexes, whereas these residues were FEBS Journal 277 (2010) 3699–3714 ª 2010 The Authors Journal compilation ª 2010 FEBS 3707 HIV protease mutants altering hydrophobic clusters A C-H Shen et al RMS differences (Å) 2.5 1.5 0.5 26 51 76 1′ Residue (#) 26′ 51′ 76′ B SQV APV 0.6–0.8 0.5–0.9 0.1–0.4 0.5–0.9 SQV APV C Asp30′ 0.4 Pro81 1.8 1.1 Pro81′ 0.6 Asp30 ˚ 0.7–2.5 A further apart in the SQV complexes (separa˚ tions of 18.5–20.5 A) The comparison of wild-type complexes is shown in Fig 4C PRI50V complexes had the smallest distance between Pro81 and Pro81¢, 3708 Fig Structural differences between APV and SQV complexes (A) The RMS differ˚ ence (A) per residue is plotted for Ca atoms of SQV complexes compared with the corresponding APV complexes: PRWT (blue line), PRI50V (red line) and PRI54V (green line) (B) Comparison of the flap regions in the structures The complexes with APV are in cyan, and the complexes with SQV are in gray The arrow indicates the shifts between Ca atoms at the residues 50 and 51 in the PR complexes with the two inhibitors (C) The width across the S1–S1¢ subsites increases in PRWT–SQV relative to PRWT–APV Similar changes were seen for the mutant complexes, except for PRI50V whereas the greatest separation was observed for PRI54M complexes, probably due to close contacts of the longer Met54 ⁄ 54¢ side chains with the 80¢s loops in both inhibitor complexes The distance between Pro81 FEBS Journal 277 (2010) 3699–3714 ª 2010 The Authors Journal compilation ª 2010 FEBS C-H Shen et al HIV protease mutants altering hydrophobic clusters ˚ and Pro81¢ in the other structures was  2.5 A longer in the SQV complexes compared with the APV complexes, which corresponds to the increment of the width across the S1 ⁄ S1¢ pockets caused by binding of the big decahydroisoquinoline P1¢ group of SQV instead of the smaller P1¢ group in APV (Fig 4C) [12] The more rigid region of Asp30 ⁄ 30¢ showed smaller ˚ shifts of  0.5 A for the Ca atoms The different P2 and P2¢ groups, THF in APV or Asn in SQV at P2 and aniline in APV or t-butyl group in SQV at P2¢, are accommodated by these shifts, as shown in Fig 4C A WT The side chain interactions within the inner hydrophobic cluster were analyzed in PRWT, PRI50V, PRI54M and PRI54V complexes with SQV and APV Overall, the main chains of the flaps were shifted relative to the 80¢ loops in APV complexes compared with SQV complexes The hydrophobic cluster around the active site was formed by Ile47, Ile54, Thr80 and Ile84 from one subunit and Ile50¢ from the opposite subunit, as well as Val32 in a more rigid region in PRWT Differences in the side chain interactions are described In PRWT–APV the Cc1 of Ile50 made good van der Waals contacts with Ile54¢, but the side chains were further B C Ile54′ I54M I50V Met54′ Ile54′ 3.8 3.9 4.1 3.9 3.9 3.8 4.2 4.0 4.1 3.8 Ile50 Val50 3.9 Ile50 3.5 3.6 3.5 3.8 3.2 3.6 Thr80′ Thr80′ Thr80’ D E I84V Leu23 I54V Val54′ 4.2 3.7 Asp25 3.5 4.2 Val82 3.6 4.1 3.7 Ile50 3.7 Val84 4.0 Ala28 3.5 4.2 3.3 4.1 Val32 Thr80′ Fig Interactions of Ile50, Ile54¢ and Thr80¢ (A) PRWT–APV compared with PRWT–SQV (B) PRI50V–APV compared with PRI50V–SQV (C) PRI54M–APV compared with PRI54M–SQV (D) PRI54V–APV compared with PRI54V–SQV (E) PRI84V–APV compared with PRI84V–SQV Dashed ˚ lines indicate van der Waals contacts with interatomic distances in A Dotted lines indicate C-HỈỈỈO interactions Black lines indicate interactions in SQV complexes Red lines indicate interactions in APV complexes FEBS Journal 277 (2010) 3699–3714 ª 2010 The Authors Journal compilation ª 2010 FEBS 3709 HIV protease mutants altering hydrophobic clusters C-H Shen et al apart in PRWT–SQV (Fig 5A) One C-HỈỈỈO interaction between Cd1 of Ile50 ⁄ Ile50¢ and the hydroxyl of Thr80¢ ⁄ Thr80 was conserved in PRWT–APV, PRWT– SQV and PRI50V–SQV The structure of PRI50V–APV, however, lost this C-HỈỈỈO interaction of residue 50 with the hydroxyl of Thr80¢, which reduced the interactions between the flap and the 80¢s loop (Fig 5B) Mutation of Thr80 to Val80, which eliminates the C-HỈỈỈO interaction with Ile50 Cd1, significantly reduces the catalytic activity and binding affinity of SQV [36] Apart from the different interactions between PR and inhibitors, the loss of the C-HỈỈỈO contact between residue 50 and Thr80 in PRI50V–APV and not in PRI50V–SQV appears to correlate with the observed drug resistance Mutation I50V is associated strongly with resistance to APV, but not to SQV In the PRI54M and PRI54V complexes with both inhibitors, however, similar interactions were observed among the side chains of residues 50, 54¢ and 80¢ (Fig 5C, D) Val84 in PRI84V–SQV had fewer hydrophobic contacts than in PRI84V–APV, but it had one C-HỈỈỈO interaction between Cc2 of Val84 and the carbonyl oxygen of Val82 (Fig 5E) In contrast to the significant conformational shifts in main chain atoms, the side chains in this hydrophobic cluster generally have rearranged to maintain internal hydrophobic contacts in the complexes with both inhibitors Discussion We have described an atomic resolution crystal structure of PRWT with APV, and analyzed structural changes in the APV complexes with mutants PRV32I, PRI50V, PRI54M, PR154V, PRI84V and PRL90M The mutated residues contribute to an inner hydrophobic cluster around the substrate binding cavity, with the exception of Leu90, which is located near the backbone of the catalytic Asp25 in the outer hydrophobic cluster (Fig 1B) Studies of the patterns of resistance mutations and molecular dynamics simulations have suggested the importance of these hydrophobic mutations in drug resistance [14,41] Our analysis showed that interactions within the inner hydrophobic cluster containing residues 32, 47, 54 and 50 were frequently altered relative to those in the wild-type enzyme Mutations to larger side chains in PRV32I, PRI54M and PRL90M resulted in the formation of new hydrophobic contacts with flap residues, residues 79 and 80, and Asp25, respectively Mutation to smaller side chains caused loss of internal hydrophobic interactions in the PRI50V and PRI54V structures PRI84V, PRV32I and PRI50V showed reduced APV inhibition by 6-, 10- and 30-fold, respectively, relative to PRWT, 3710 which is consistent with the observed structural changes The PRI84V–APV complex had lost hydrophobic contacts with APV, the PRV32I–APV complex showed increased hydrophobic contacts with the flaps that probably restricted the flexibility needed for catalysis, and the PRI50V complex had weaker interactions with APV Ile54 had no direct contact with APV, which is consistent with the relatively small changes in inhibition, catalytic properties, protease stability and structure shown by the I54 mutants No compensating changes were identified elsewhere in the hydrophobic core In PRL90M, the longer side chain of Met90 ⁄ 90¢ lies close to the main chain of the catalytic aspartates forming new van der Waals contacts and a C-HỈỈỈO interaction These new contacts with Asp25 ⁄ 25’ near the dimer interface correlate with the reduced stability and altered catalytic parameters of this mutant, as described in studies with indinavir and DRV [19,25] No evidence was found, however, that PRL90M–APV had substantially altered the volume of the S1 ⁄ S1¢ substrate binding pockets, unlike the PRG48V ⁄ L90M–SQV structure, which showed reduced volume for the S1 ⁄ S1¢ subsites relative to the wildtype complex [37] Also, the structure of PRG48V– DRV showed reduced volume of the active site cavity relative to the wild-type complex, consistent with a major effect of the G48V rather than the L90M mutation in reducing the S1 ⁄ S1¢ volume in PRG48V ⁄ L90M– SQV [18] Reduced interactions with inhibitors and conformational adjustments of the flaps and 80¢s loops were observed in our previous studies of these mutants with other inhibitors [17–19,25,30,42] These structural changes are expected to contribute to drug resistance However, crystal structures show only a static picture of the effect of mutations, whereas changes in protein dynamic and thermodynamic properties also probably contribute to resistance Future studies using molecular dynamics simulations and calorimetric analysis will help to address the changes in these other properties Individual mutations have distinct effects on the protease structure and activity However, drug-resistant clinical isolates generally accumulate multiple mutations Previous studies of double and single mutants suggested that the structural changes due to a single mutation were retained in the double mutant, although other properties did not combine in predictable ways [43] A variety of structural and biochemical mechanisms have been reported for different combinations of mutations, including: compensating structural changes, altered interactions with inhibitor and substantial opening of the active site cavity [44–46] Clearly, further studies are needed to assess the effects FEBS Journal 277 (2010) 3699–3714 ª 2010 The Authors Journal compilation ª 2010 FEBS C-H Shen et al of the multiple protease mutations that are observed clinically Comparison of the APV complexes with the corresponding SQV complexes for PRWT, PRI50V, PRI54V and PRI84V showed changes in the conformation of the flexible flaps and 80¢s loops Despite the conformational changes in main chain atoms, the internal side chains generally rearranged to preserve the internal hydrophobic contacts in the complexes with both inhibitors The structural changes can be correlated with the type of inhibitor In particular, the separation of Pro81 and Pro81¢ was significantly smaller in the complexes with APV compared with the equivalent SQV complexes, which reflects the smaller size of the P1¢ group in APV relative to that of SQV Also, the flexible side chains of Asp30 and Asp30¢ accommodate diverse functional groups at P2 and P2¢ of SQV and APV at the surface of the PR active site cavity The functional group can be critical for a tight binding inhibitor For example, DRV was derived from APV by changing THF to bis-THF, which introduces more hydrogen bonds with PR main chain atoms and dramatically increases the potency on drug-resistant HIV [47] APV was designed to include several hydrophilic interactions with PR, and SQV optimizes hydrophobic interactions with PR [12] Both of the inhibitors have been classified as peptidomimetic inhibitors, which mimic PR–substrate interactions and block enzyme activity, although APV has only a single CO-NH peptide bond compared with three in SQV [48] Resistant mutations within the hydrophobic clusters frequently involve small changes such as the addition or deletion of a methylene group [13] Mutations within the hydrophobic cluster have the potential to alter the flap dynamics or stability of PR as well as the binding of inhibitors, as shown in our structural analysis The current drugs target the active site cavity and demonstrate strong binding to the catalytic Asps [49] The hydrophobic pockets around the substrate binding site have been proposed as an alternative drug target [50,51] However, our structural analysis showed that side chains in the hydrophobic cluster can rearrange readily to maintain the PR structure and activity, suggesting that this region is a poor target for drugs The accuracy of high and atomic resolution crystal structures is critical for deciphering how mutations and inhibitors alter the PR structure Here, we describe how PR recognizes the inhibitors APV and SQV by structural rearrangements of the two beta-hairpin flaps and two 80¢s loops, and how the same mutation results in different structural changes with the two drugs Comparison of the structures provides insight into why I50V is a major drug-resistant mutation observed HIV protease mutants altering hydrophobic clusters on exposure to APV, but appears less critical in resistance to SQV therapy Apart from the different interactions between PR and inhibitors, the absence of the C-HỈỈỈO contact between flap residue 50 and Thr80 in PRI50V–APV, and its presence in PRI50V–SQV and PRWT complexes, contributes to the drug resistance of this mutation The conclusion is that small rearrangements of the PR loops enclosing the inhibitor combined with changes in weak internal interactions produce the distinct patterns of resistant mutations for the two drugs Experimental procedures Protein expression and purification PR and mutants were constructed with five mutations Q7K, L33I, L63I, C67A and C95A to prevent cysteine-thiol oxidation and diminish autoproteolysis [52] The expression, purification and refolding methods are described in [52,53] Kinetic assays The fluorogenic substrate Abz-Thr-Ile-Nle-p-nitro-Phe-GlnArg-NH2, where Abz is anthranilic acid and Nle is norleucine (Bachem, King of Prussia, PA, USA), with the sequence derived from the p2 ⁄ NC cleavage site of the Gag polyprotein, was used in the kinetic assays Proteases were diluted in reaction buffer (100 mm Mes, pH 5.6, 400 mm sodium chloride, mm EDTA and 5% glycerol) Ten microliters of diluted enzyme was mixed with 98 lL reaction buffer and lL dimethylsulfoxide or APV (dissolved in dimethylsulfoxide) and incubated at 37 °C for The reaction was then initialized with the addition of 90 lL substrate The reaction was monitored over in the POLARstar OPTIMA microplate reader at wavelengths of 340 and 420 nm for excitation and emission Data analysis used the program sigmaplot 9.0 (SPSS Inc., Chicago, IL, USA) Km and kcat values were obtained by standard data fitting with the Michaelis–Menten equation The Ki value was obtained from the IC50 values estimated from an inhibitor dose–response curve using the equation Ki = (IC50 ) [E] ⁄ 2) ⁄ (1 + [S] ⁄ Km), where [E] and [S] are the PR and substrate concentrations Crystallographic analysis Inhibitor APV (from the AIDS reagent program) was dissolved in dimethylsulfoxide by vortex mixing The mixture was incubated on ice prior to centrifugation to remove any insoluble material The inhibitor was mixed with 2.2 mgỈmL)1 protein in a molar ratio of : in most cases The two exceptions were: 3.5 mgỈmL)1 PRI50V was used with an inhibitor ⁄ protein ratio of 10:1, and 3.7 mgỈmL)1 FEBS Journal 277 (2010) 3699–3714 ª 2010 The Authors Journal compilation ª 2010 FEBS 3711 HIV protease mutants altering hydrophobic clusters C-H Shen et al PRL90M The crystallization trials employed the hanging drop vapor diffusion method using equal volumes of enzyme–inhibitor and reservoir solution PRWT–APV was crystallized from 0.1 m Mes, pH 5.6 and 0.6–0.8 m sodium chloride Crystals of PRV32I–APV and PRI50V–APV were grown from 0.1 m sodium acetate, pH 5.4, 0.4 and 1.2 m sodium chloride, respectively PRI54M–APV crystals were grown from 0.1 m sodium acetate, pH 4.6 and 0.67 m sodium chloride PRI54V–APV and PRI84V–APV crystals were grown from 0.1 m sodium acetate, pH 5.4 and 4.0, respectively, and 0.13 m sodium iodide, and PRL90M–APV crystals from 0.1 m sodium acetate, pH 4.8 and 0.2 m sodium iodide Single crystals were mounted on fiber loops with 20–30% (v ⁄ v) glycerol as the cryoprotectant in the reservoir solution X-ray diffraction data were collected at the SER-CAT beamline of the Advanced Photon Source (Argonne National Laboratory, Argonne, IL, USA) Diffraction data were integrated, scaled and merged using the HKL2000 package [54] PRWT–APV, PRV32I–APV and PRI50V–APV were solved by the molecular replacement program phaser [55] with the protein atoms of structure 2QCI [32] as the starting model The other complexes were solved by molrep [56], using the protein atoms of 2F8G as the starting model [19] The crystal structures were refined using shelx-97 [57], except that the lower resolution structure of PRI84V–APV was refined with refmac 5.2 [58] DPI was used for determining the accuracy in the atomic positions [59] The molecular graphics program coot was used for map display and model building [60] Structural figures were made by pymol [61] The structures were compared by superimposing their Ca atoms and using hivagent [62] to calculate the distance between two atoms The cut-off distances for different interactions were as described in [30] Acknowledgements CS was supported in part by the Georgia State University Research Program Enhancement award The research was supported in part by the National Institutes of Health grant GM062920 We thank the staff at SER-CAT beamline at the Advanced Photon Source, Argonne National Laboratory, Argonne, IL, USA, for assistance during X-ray data collection Use of the Advanced Photon Source was 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material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 3699–3714 ª 2010 The Authors Journal compilation ª 2010 FEBS ... PRI54M, PRI54V, PRI84V and PRL90M complexes with APV Table S1 Hydrogen bond interactions between HIV-1 protease and APV Table S2 C-HỈỈỈO interactions between HIV-1 protease and APV This supplementary... interaction and weaker interactions than the major conformation had with PR Effects of mutations on PR structure and interactions with APV The structures of the mutants and PRWT complexes with APV... the volume of the active site cavity and alter the hydrophobic interactions in the cluster HIV protease mutants altering hydrophobic clusters The mutant with Ile32 did not show significant alterations